Purification of 5 beta-reductase from hepatic cytosol fraction of chicken.
From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5 beta-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5 beta-reduced metabolites derived from [4-14C]testosterone. During the purification procedures, 17 beta-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5 beta-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were able to be removed from 5 beta-reductase fraction. The final enzyme preparation was apparently homogeneous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5 beta-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. Km of 5 beta-reductase for NADPH was estimated as 4.22 x 10(-6) M and for testosterone, 4.60 x 10(-6) M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5 beta-reductase were examined.
Sugimoto
Y
Y
Department of Radiopharmacology, Faculty of Pharmaceutical Sciences, Nagasaki University, Japan.
Yoshida
M
M
Tamaoki
B
B
D000645:Ammonium Sulfate; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D002645:Chickens; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification*; D011374:Progesterone / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
D013739:Testosterone; D000735:Androstenedione; D011374:Progesterone; D009249:NADP; D010088:Oxidoreductases; C051860:cholestenone 5 beta-reductase; D000645:Ammonium Sulfate